PCR and sequencing

Question 1

Components of Sanger sequencing?

Answer 1

• DNA template
• sequencing primer
• thermostable DNA polymerase
• nucleotides (dNTPs)
• dideoxynucleotides (ddNTPs)
• buffer

Question 2

Descibe the process of Sanger sequencing

Answer 2

Steps
1. Chain-termination PCR
- use a low ratio of chain-terminating ddNTPs. ddNTPs lack the 3’-OH group required for phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension ceases. The result of chain-termination PCR is millions to billions of oligonucleotide copies of the DNA sequence of interest, terminated at a random lengths.

2. Size separation by gel Gel Electrophoresis
   - In automated Sanger sequencing, all oligonucleotides are run in a single capillary gel electrophoresis within the sequencing machine.
3. Read DNA
   In automated Sanger sequencing, a computer reads each band of the capillary gel, in order, using fluorescence to call the identity of each terminal ddNTP. In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light emitted. Because each of the four ddNTPs is tagged with a different fluorescent label, the light emitted can be directly tied to the identity of the terminal ddNTP. The output is called a chromatogram, which shows the fluorescent peak of each nucleotide along the length of the template DNA.

Question 3

Components of PCR?

Answer 3

• Template DNA.
• DNA polymerase (Taq)
• Primers: Forward and Reverse
• Deoxynucleoside riphosphates (dNTPs)
• Buffer which:
  Stabilizes the DNA polymerase, DNA, and nucleotides and contains MgCl2

Question 4

We start with three molecules of the template DNA. How many molecules will we have after 25 PCR cycles?

Answer 4

3 x 225 = 100,663,296

In general, if we start with N molecules of the template DNA and perform n
PCR cycles, we will end up with
N x 2n
molecules of amplified DNA

Question 5

What is RT-PCR?

Answer 5

A PCR
variant that starts
from RNA
template

Question 6

PCR applications?

Answer 6

Molecular cloning
* Labelling of DNA fragments
* Genetic tests (rare diseases and forensic applications)
* Oncological diagnostics
* Infection disease diagnostics (SARS-CoV-2, HIV etc.)

Question 7

What is the annealing temperature for the primer:
TCGAATCGATAGCTTCGGA

Answer 7

Tm = 4oC (G+C) + 2oC (A+T)
Tm = 56
Tann=Tm - 5oC
Tann = 51